Traumatic injury of the spinal cord results in permanent functional impairment. Most of the deficits associated with spinal cord injury result from the loss of axons that are damaged in the central nervous system (CNS). Similarly, other diseases of the CNS are associated with axonal loss and retraction, such as stroke, human immunodeficiency virus (HIV), dementia, prion diseases, Parkinson's disease, Alzheimer's disease, multiple sclerosis, traumatic brain injury, macular degeneration and glaucoma. Common to all of these CNS diseases, including many of the eye diseases, is the loss of axonal connections with their targets, and cell death. The ability to stimulate growth of axons from the affected or diseased neuronal population would improve recovery of lost neurological functions, and protection from cell death can limit the extent of damage. For example, following a white matter stroke, axons are damaged and lost, even though the neuronal cell bodies are alive, and stroke in grey matter kills many neurons and non-neuronal (glial) cells. Treatments that are effective in eliciting sprouting from injured axons are equally effective in treating some types of stroke (Chen et al., 2002, PNAS 99:9031-9036). Neuroprotective agents are often tested as potential compounds that can limit damage after stroke. Compounds which show both growth-promotion and neuroprotection are especially good candidates for treatment of stroke and neurodegenerative diseases.
It has been proposed to use various Rho antagonists as agents to stimulate regeneration of (cut) axons, i.e. nerve lesions; please see, for example, Canadian Patent application Nos 2,304,981 (McKerracher et al.) and 2,300,878 (Strittmatter). These patent application documents propose the use of known Rho antagonists such as, for example, the chimeric C3 proteins as well as substances selected from among known trans-4-amino (alkyl)-1-pyridylcarbamoylcyclohexane compounds or Rho kinase inhibitors for use in the regeneration of axons. C3 inactivates Rho by ADP-ribosylation and is fairly non-toxic to cells (Dillon and Feig, 1995, Methods in Enzymology: Small GTPases and their regulators Part. B, 256: 174-184).
Treatment with Rho antagonists would also be used to enhance the rate of axon growth of peripheral nerves and thereby be effective for repair of peripheral nerves after surgery, for example after reattaching severed limbs. In addition, Rho is an important target for treatment of cancer and metastasis (Clark et al., 2000, Nature, 406: 532-535), and hypertension (Uehata et al., 1997, Nature, 389: 990) and RhoA is reported to have a cardioprotective role (Lee et al., FASEB J., 15: 1886-1884).
Targeting intracellular signaling mechanisms involving Rho and the Rho kinase for promoting axon regeneration has been proposed (Canadian Patent application No 2,304,981). Clostridium botulinum C3 exotransferase (hereinafter simply referred to as C3) can stimulate regeneration and sprouting of injured axons; C3 is a toxin purified from Clostridium botulinum (Wilde et al., 2000, J. Biol. Chem., 275: 16478). Compounds of the C3 family from Clostridium botulinum inactivate Rho by ADP-ribosylation and thus act as antagonists of Rho effect or function (Rho antagonists).
While the C3 protein can effectively promote regeneration, it has been noted that C3 does not easily penetrate into cells, and high doses must therefore be applied for it to be effective. The high dose of recombinant C3 needed to promote functional recovery presents a practical constraint or limitation on the use of C3 in vivo to promote regeneration. In tissue culture studies, it has been determined that the minimum amount of C3 that can be used to induce growth on inhibitory substrates is 25 μg/ml (Lehmann et al., 1999, J. Neurosci. 19: 7537-7547). If the cells are not triturated, even this dose is ineffective. It has been estimated that at least 40 μg of C3 per 20 g of mouse needs to be applied to injured mouse spinal cord or rat optic nerve (Canadian patent application No 2,325,842). Calculating doses that would be required to treat an adult human with an equivalent dose per weight (scaling up from the dose used for rat and mice experiments) it would be necessary to apply 120 mg/kg of C3 to the injured human spinal cord. The large amount of recombinant C3 protein needed creates significant problems for manufacturing, due to the large-scale protein purification and cost. It also limits the dose ranges that can be tested because of the large amount of protein needed for minimal effective doses.
Another related limitation with respect to the use of C3 to promote repair in the injured CNS is that it does not easily penetrate the plasma membrane of living cells. In tissue culture studies where C3's biological effects have been tested, it was microinjected directly into the cell (Ridley and Hall, 1992, Cell, 70: 389-399), or applied by trituration of the cells to break the plasma membrane (Lehmann et al., 1999, J. Neurosci., 19: 7537-7547). In the case of axon injury in vivo, the C3 protein is likely able to enter the cell because injured axons readily take up substances from their environment. After incomplete spinal cord injury (SCI), there is plasticity of motor systems attributed to cortical and subcortical levels, including spinal cord circuitry. This plasticity may be attributed to axonal or dendritic sprouting of collaterals and synaptic strengthening or weakening. Additionally, it has been shown that sparing of a few ventrolateral fibers may translate into significant differences in locomotor performance since these fibers are important in the initiation and control of locomotor pattern through spinal central pattern generators. It is well documented that reorganization of spared collateral cortical spinal fibers occurs after spinal cord injury and this contributes to functional recovery. The process of reorganization and sprouting of spared fibers would be enhanced by treatment with C3-like proteins able to enter non-injured neurons. This would enhance spontaneous plasticity of axons and dendritic remodeling known to help functional recovery.
Other methods of delivery of C3 in vitro include making a recombinant protein that can be taken up by a receptor-mediated mechanism (Boquet et al., 1995, Meth. Enzymol., 256: 297-306). The disadvantage of this method is that the cells needing treatment must express the necessary receptor. Lastly, addition of a C2II binding protein to the tissue culture medium, along with a C21N-C3 fusion toxin allows uptake of C3 by receptor-mediated endocytosis (Barthe et al., 1998, Infection and Immunity, 66: 1364). The disadvantage of this system is that much of the C3 in the cell will be restrained within a membrane compartment. More importantly, two different proteins must be added separately for transport to occur, which makes this system difficult to apply to treatment of disease in vivo.
Currently, there is a need to find a therapy that can stop degenerative progression in people who have eye diseases. The neurons of the retina are derived from the CNS, and also are expected to respond to treatments effective in other regions of the CNS, for example, age-related macular degeneration (AMD). Most experimental forms of treatment known to date address the wet form of AMD, and target specifically neovascularization. Laser photocoagulation of the subretinal neovascular membranes that occur in 10-15% of affected patients can benefit individuals with macular degeneration who develop acute, extrafoveal choroidal neovascularization. For dry AMD, high daily doses of antioxidants such vitamin C (500 mg), vitamin E (400 IU), beta carotene (15 mg), as well as zinc oxide (80 mg; high concentrations of zinc occur in ocular tissues, particularly the retina, pigment epithelium and choroid) may modestly reduce risk of progression of those who have intermediate-sized drusen, large drusen, or non-central geographic atrophy, or advanced macular degeneration in one eye. There is current need of therapy to treat such eye diseases with compounds that protect the retinal neurons. There is also a current need of therapy for persons with acute ocular ischemic disease. Ocular ischemic disease, or stroke of the optic nerve results in irreversible death of retinal neurons, leading to permanent visual impairment. This disease is not expected to respond to current therapies for AMD. C3-like proteins may reduce the cell death and progression of the disease.
Therefore, the new C3-like proteins are expected to be useful for a variety of diseases where inhibition of Rho activity is required. Thus, there is a need for compounds, methods of treatment and formulations to treat or prevent diseases where inhibition of Rho activity is required. It would also be desirable to be provided with C3-like protein compositions having the ability to penetrate inside tumor cells and inactivate rapidly Rho at lower doses.